Bacteriophage amplification has been suggested as a method to accelerate microorganism identification. See, for example, U.S. Pat. No. 5,985,596 issued Nov. 16, 1999 and U.S. Pat. No. 6,461,833 B1 issued October 8, both to Stuart Mark Wilson; U.S. Pat. No. 4,861,709 issued Aug. 29, 1989 to Ulitzur et al.; U.S. Pat. No. 5,824,468 issued Oct. 20, 1998 to Scherer et al.; U.S. Pat. No. 5,656,424 issued Aug. 12, 1997 to Jurgensen et al.; U.S. Pat. No. 6,300,061 B1 issued Oct. 9, 2001 to Jacobs, Jr. et al.; U.S. Pat. No. 6,555,312 B1 issued Apr. 29, 2003 to Hiroshi Nakayama; U.S. Pat. No. 6,544,729 B2 issued Apr. 8, 2003 to Sayler et al.; U.S. Pat. No. 5,888,725 issued Mar. 30, 1999 to Michael F. Sanders; U.S. Pat. No. 6,436,661 B1 issued Aug. 20, 2002 to Adams et al.; U.S. Pat. No. 5,498,525 issued Mar. 12, 1996 to Rees et al.; Angelo J. Madonna, Sheila VanCuyk and Kent J. Voorhees, “Detection Of Escherichia Coli Using Immunomagnetic Separation And Bacteriophage Amplification Coupled With Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry”, Wiley InterScience, DOI:10.1002/rem.900, 24 Dec. 2002; and U. S. Patent Application Publication No. 2004/0224359 published Nov. 11, 2004, Bacteriophage are viruses that have evolved in nature to use bacteria as a means of replicating themselves. A bacteriophage (or phage) does this by attaching itself to a bacterium and injecting its genetic material into that bacterium, inducing it to replicate the phage from tens to thousands of times. Some bacteriophage, called lytic bacteriophage, rupture the host bacterium releasing the progeny phage into the environment to seek out other bacteria. The total incubation time for infection of a bacterium by parent phage, phage multiplication (amplification) in the bacterium to produce progeny phage, and release of the progeny phage after lysis can take as little as an hour depending on the phage, the bacterium, and the environmental conditions. Thus, it has been proposed that the use of bacteriophage amplification in combination with a test for bacteriophage or a bacteriophage marker may be able to significantly shorten the assay time as compared to a traditional substrate-based identification. A single infected bacterium may produce 101-104 progeny bacteriophage, and each bacteriophage particle may contain 101-103 copies of capsid or other structural proteins. Signal amplifications of 102-107 from each infected bacterium, therefore, are possible, given an appropriate method of detecting progeny bacteriophage, bacteriophage nucleic acids, or bacteriophage proteins. Many methods known to the art are suitable for detection, including but not limited to, PCR, mass spectrometry, antibody or aptamer-based binding assays, and plaque assays.
In each of the bacteriophage amplification methods mentioned above, samples potentially containing target bacteria are incubated with bacteriophage specific for those bacteria. In the presence of the bacteria, the bacteriophage infect and replicate in the bacteria resulting in the production of a measurable signal indicating the presence of the target bacteria. Some methods utilize the detection of progeny phage released from infected target bacteria as a means of detection and identification. In this case, progeny phage are not produced if the parent phage do not successfully infect the target bacteria. Still other methods rely on the detection of phage replication products rather than whole progeny phage. For example, luciferase reporter bacteriophage produce luciferase when they successfully infect target bacteria. The luciferase then produces light that, if detected, indicates the presence of target bacteria in the sample. Other methods rely on the detection of bacterial debris that is released following a successful lytic infection of target bacteria by a specific bacteriophage. Still other methods rely only on the ability of bacteriophage to attach to the bacteria and do not employ amplification. To accurately identify the target bacteria, each of these phage-based diagnostic methods demands that the bacteriophage have both high sensitivity for the target bacteria and high specificity to avoid replication in non-target strains or species of bacteria. Finding or developing bacteriophage with those characteristics is very challenging. Thus, while bacteriophage amplification is considered as a promising process for detecting microorganisms, a commercially useful diagnostic process using bacteriophage that is competitive with conventional commercial microorganism detection processes has not yet been developed. Bacteriophage with acceptable sensitivity often lack sufficient specificity, i.e., they cross react with too many non-target bacteria. This lack of acceptable sensitivity in combination with sufficient specificity is a critical problem in commercializing bacteriophage diagnostic processes.
It is well known that, within a given bacterial species, individual strains vary in their susceptibility to bacteriophage strains; in fact, this differential susceptibility forms the basis of phage-typing schemes for the identification of bacterial strains. The biochemical basis of this differential susceptibility is not well understood, but some factors have been identified. These include virulence factors, often found on mobile genetic elements within bacterial chromosomes. A well-known example is the Staphylococcus aureus (S. aureus) factor for Toxic Shock Syndrome, encoded by the pathogenicity island SaPI1, found in approximately 20% of clinical isolates of S. aureus. These pathogenicity islands are mobilized by infection of the host bacterium by bacteriophages and are encapsidated into infectious particles. This mobilization and encapsidation takes place at the expense of the infecting bacteriophage, whose replication can be reduced by a factor of 100 fold (Lindsay et al., Molecular Microbiology, 1998, 29:2527). This reduction is problematic for any assay or process dependent upon bacteriophage amplification, as it renders a substantial fraction of bacterial hosts incapable of producing high bacteriophage yields.
Thus, there remains a need for a faster and more effective method of detecting microorganisms that achieves both specificity and sensitivity and, at the same time, the amplification remains high.